Figure 1.

Costunolide induces glioma cell death and decreases telomerase activity in ROS/p53-dependent manner. (a) Costunolide reduces viability of A172 and U87MG glioma cells, but has no effect on p53 mutant T98G cells. Graph shows percent change in viability upon treatment with different doses of Costunolide for 24 h as determined by MTS assay. (b) The increase in DHE fluorescence induced upon Costunolide treatment is abrogated in the presence of ROS inhibitor NAc. (c) Costunolide-induced increase in TUNEL-positive cells is abrogated in the presence of NAc. Graph depicts the percent of TUNEL-positive cells treated with Costunolide, NAc or both. (d) ROS inhibition rescues Costunolide-mediated decrease in telomerase activity. Graph shows fold change in telomerse activity over control in glioma cells treated with different combinations of Costunolide and NAc. (e) Overexpression of SOD-1 rescues Costunolide-mediated decrease in hTERT expression. Following transfection of glioma cells with SOD-1 overexpression construct for 48 h, cells were treated in the presence and absence of Costunolide for an additional 24 h and hTERT levels were determined. Inset demonstrates SOD-1 expression in transfected cells. (f) Costunolide-induced increase in p53 expression is ROS dependent. Western blot images depicting p53 levels in cells treated with Costunolide in the presence or absence of NAc. (g) siRNA-mediated knock-down of p53 prevents Costunolide-mediated decrease in telomerase activity. Graph represents the telomerase activity of glioma cells transfected with either p53 siRNA or scrambled (NS) siRNA and treated with Costunolide. Values are expressed as fold change over control. Inset confirming the transfection efficiency of p53 siRNA. Values in the graph (a–d and g) represent the means±S.E.M. from three independent experiments. *Significant change from control, ^#Significant change from Costunolide-treated cells (P<0.05). Blots (e and f) are representative images of three independent experiments showing similar results. Blots were re-probed for c23 to establish equivalent loading